Wang et al. BMC Cancer (2019) 19:1258 https://doi.org/10.1186/s12885-019-6484-5

RESEARCH ARTICLE Open Access

Downregulated SPINK4 is associated with

poor survival in colorectal cancer Xiaojie Wang1†, Qian Yu2†, Waleed M. Ghareeb1,3, Yiyi Zhang1, Xingrong Lu1, Ying Huang1, Shenghui Huang1,Yanwu Sun1, Jiayi Lin4, Jin Liu4 and Pan Chi1*

Abstract

Background: SPINK4 is known as a gastrointestinal peptide in the gastrointestinal tract and is abundantlyexpressed in human goblet cells. The clinical significance of SPINK4 in colorectal cancer (CRC) is largely unknown.

Methods: We retrieved the expression data of 1168 CRC patients from 3 Gene Expression Omnibus (GEO) datasets(GSE24551, GSE39582, GSE32323) and The Cancer Genome Atlas (TCGA) to compare the expression level of SPINK4between CRC tissues and normal colorectal tissues and to evaluate its value in predicting the survival of CRCpatients. At the protein level, these results were further confirmed by data mining in the Human Protein Atlas andby immunohistochemical staining of samples from 81 CRC cases in our own center.

Results: SPINK4 expression was downregulated in CRC compared with that in normal tissues, and decreasedSPINK4 expression at both the mRNA and protein levels was associated with poor prognosis in CRC patients fromall 3 GEO datasets, the TCGA database and our cohort. Additionally, lower SPINK4 expression was significantlyrelated to higher TNM stage. Moreover, in multivariate regression, SPINK4 was confirmed as an independentindicator of poor survival in CRC patients in all databases and in our own cohort.

Conclusions: We concluded that reduced expression of SPINK4 relates to poor survival in CRC, functioning as anovel indicator.

Keywords: Colorectal cancer, SPINK4, Survival

Highlight

� SPINK4 expression was downregulated in colorectalcancer compared with that in normal tissues.

� Decreased SPINK4 mRNA expression was associatedwith poor prognosis in colorectal cancer patients in3 independent databases (GSE24551, GSE39582, theTCGA database) and our own cohort.

� This study is the first, to the best of our knowledge,to implicate SPINK4 as a novel indicator of poorsurvival in CRC.

BackgroundDespite significant progress in surgery, radiotherapy,chemotherapy, and targeted therapy, colorectal cancer

© The Author(s). 2019 Open Access This articInternational License (http://creativecommonsreproduction in any medium, provided you gthe Creative Commons license, and indicate if(http://creativecommons.org/publicdomain/ze

* Correspondence: chipan363@163.com†Xiaojie Wang and Qian Yu contributed equally to this work.1Department of Colorectal Surgery, Union Hospital, Fujian Medical University,29 Xin-Quan Road, Fuzhou, Fujian 350001, People’s Republic of ChinaFull list of author information is available at the end of the article

(CRC) remains one of the leading cancer types in termsof incidence and cancer-related death worldwide [1].This characteristic is partly due to a lack of diagnosticmarkers for the detection of CRC and inefficient treat-ment of late-stage colorectal cancer [1]. Currently, theprediction of survival or relapse and the determinationof therapeutic strategies are mostly based on the tumor-node-metastasis (TNM) system [2]. However, the long-term outcome varies widely, even in patients within thesame TNM stage [3]. Moreover, this pathological prog-nostic prediction method alone may not accuratelypredict prognosis without incorporating molecular dataof the tumor [4]. Hence, an increasing number of studiesin this era of genomic medicine have focused on mo-lecularly based prognostic markers, which are comple-mentary to the pathological TNM system [5, 6].Serine protease inhibitors function as central regulators

of many vital processes in the mammalian body; whenserine protease activity or serpin-mediated regulation

le is distributed under the terms of the Creative Commons Attribution 4.0.org/licenses/by/4.0/), which permits unrestricted use, distribution, andive appropriate credit to the original author(s) and the source, provide a link tochanges were made. The Creative Commons Public Domain Dedication waiverro/1.0/) applies to the data made available in this article, unless otherwise stated.

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becomes unbalanced or dysfunctional, severe diseasestates, such as cancer and sepsis, can ensue [7]. Onebranch of the family of serine protease inhibitors is namedKazal type (SPINK) and originally consisted of four mem-bers in humans (SPINK1, SPINK2, SPINK4, and SPINK5)[8]. Although the major site of expression of all fourSPINK members may differ, all are thought to be involvedin protection against proteolytic degradation of epithelialand mucosal tissues [8]. SPINK4 is abundantly expressedin human goblet cells but was also reported to be formed,stored, and secreted from monocytes and might functionas a gastrointestinal peptide [9]. A previous study foundthat serum SPINK4 levels were increased in CRC and hadhigh diagnostic value but were not associated with thesurvival of CRC patients [10]. The expression status ofSPINK4 in tissue samples and its clinical significance inCRC is largely unknown. Therefore, the present studyaimed to measure SPINK4 expression in CRC tissues andto investigate its relationship with clinicopathological fea-tures and survival.

MethodsDatabase analysisA total of four microarray data sets were retrieved fromthe Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/). The microarray data setGSE39582 included mRNA expression profiles of a largeseries of 443 CRC and 19 nontumor colorectal mucosaand was submitted by Nabila Elarouci et al. [11]. Themicroarray data set GSE24551 comprised two independ-ent series including exon level expression profiling datafor a total of 160 CRC tissue samples and was submittedby Anita Sveen et al. [12]. The microarray data setGSE32323 included mRNA expression profiles of 17 pairsof cancer and noncancerous tissues from colorectal cancerpatients and was submitted by Kaoru Mogushi et al. [13].The majority of colorectal cancers develop as tubular ad-enomas through multistage carcinogenesis. To address thesequential expression changes in normal colonic mucosa,adenoma, and carcinoma tissues, the mRNA expressionprofiles of 4 pairs of normal colonic mucosa and adenomatissues and 4 pairs of adenoma and carcinoma tissuesfrom GSE3880 were also downloaded.The expression level of the SPINK4 gene in other cell

lines, organs and cancers was identified in the MediSapiensIST Online database (http://ist.medisapiens.com/) and theonline database Gene Expression Profiling Interactive Ana-lysis (GEPIA) (http://gepia.cancer-pku.cn/index.html) [14].RNA sequencing (RNA-Seq) data and the full clinico-

pathological dataset from 438 colon cancer patients fromCOAD were obtained from the TCGA data portal(https://portal.gdc.cancer.gov/). We excluded cases with-out sufficient survival data (n = 2), leaving 436 coloncancer patients selected for further survival analysis.

To further address the change in SPINK4 proteinexpression in CRC tissues, SPINK4 expression in CRCtissues and normal colon tissues was first reviewed byusing the immunohistochemical (IHC) staining data pro-vided in the Human Protein Atlas (http://www.proteina-tlas.org/) [15].Single-cell sequencing data and corresponding single-

cell functional states from GSE81861 [16], which in-cluded the RNA expression profiles of 44 single CRCcells, were downloaded from CancerSEA [17].A workflow of this study is shown in Fig. 1.

Tissues and relevant clinicopathological information ofpatientsA total of 81 paraffin-embedded colorectal cancer tissuesand paired adjacent paraffin-embedded normal tissueswere retrospectively collected to perform IHC staining.All patients had received radical surgery in the Depart-ment of Colorectal Surgery, Union Hospital, Fujian Med-ical University, between February 2012 and December2013. The postsurgical pathologic diagnosis in all patientswas adenocarcinoma. None of the patients received pre-operative chemotherapy or radiotherapy. The exclusioncriterion was the presence of other synchronous or meta-chronous tumors.Tissue specimens were fixed in formalin and embed-

ded in paraffin. All patients were followed up until May2018. Detailed information on the clinical characteristicsof all patients, including gender, age, body mass index(BMI), TNM stage, tumor size, histological type, pre-treatment CEA level, pretreatment CA199 level, perineu-ral invasion status, venous invasion status, tumorlocation, and tumor differentiation, was retrieved. Diag-nosis and TNM staging were performed according tothe 7th edition of the AJCC Cancer Staging Manual [18].

IHC staining and interpretation of resultsThe differential protein expression levels of SPINK4 in81 colorectal cancer and paired normal tissues weremeasured using IHC staining. Anti-SPINK4 monoclonalantibody (ab121257, Abcam, UK) was used at a workingconcentration of 1:200. The scores were evaluated basedon staining intensity and the percentage of positive cellsfor each of the sections. The staining intensity wasscored as follows: 0, no staining; 1, light yellow staining;2, yellow-brown staining; and 3, deep brown staining.The percentage of positive cells was scored as follows: 0,0~5%; 1, 6~25%; 2, 26~50%; 3, 51~75%; and 4, > 75%.The final score was calculated as follows: positive cellscore × staining intensity score. The total scores werecondensed into four categories: 0 for negative (−); 1–3for weakly positive (+); 4–7 for positive (++); and 8–12for strongly positive (+++). All patients were sorted intotwo groups according to the total score. High expression

Fig. 1 The workflow of this study

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of SPINK4 was defined as a detectable immunoreactionwith a total score of ≥1 + .

Gene set enrichment analysis (GSEA)To determine the function of SPINK4, GSEA was con-ducted in patients with the top 25% and with the bottom25% of the expression in the GSE24551 dataset andGSE39582 dataset, respectively. The annotated gene setc2.cp.kegg.v5.2.symbols.gmt from the pathway databasewas selected as the reference gene set. P < 0.05, |enrich-ment score (ES) | > 0.3 and gene size ≥30 were set as thecutoff criteria. The overlapping enriched hallmark signa-tures in the GSE24551 dataset and GSE39582 datasetare illustrated by a Venn diagram.

Statistical analysisCategorical variables were compared using the χ2 testand Fisher’s exact test. Continuous variables were com-pared using Student’s t-test. The cutoff value for SPINK4expression was assessed using X-tile 3.6.1 software (YaleUniversity, New Haven, CT, USA) [19]. Survival curveswere computed with the Kaplan-Meier method and com-pared using the log-rank test. Univariate Cox proportionalhazards regression was applied to estimate the individualhazard ratios (HRs) for the survival rates. Significantvariables in the univariate analysis (P < 0.05) were thenretained in multivariate analysis using Cox proportionalregression models to explore the independent indicators.To comprehensively explore which functional states areassociated with SPINK4 at the single-cell level, a linearmodel was used to evaluate linear correlations betweenSPINK4 expression and 14 cancer-related functional states

(stemness, invasion, metastasis, proliferation, EMT, angio-genesis, apoptosis, cell cycle, differentiation, DNA damage,DNA repair, hypoxia, inflammation and quiescence). A P-value of < 0.05 was set as the level of significance. All statis-tical analyses were performed using SPSS software (ver. 17,SPSS Inc., Chicago, IL, USA) and R (ver. 3.4.1).

ResultsThe mRNA expression of SPINK4 is downregulated in CRCtissuesTo examine the levels of SPINK4 mRNA in CRC sam-ples, we first analyzed SPINK4 mRNA expression bycomparing 17 CRC tissues and paired adjacent normaltissues from the GSE32323 dataset, and the results indi-cated that the relative SPINK4 expression level was sig-nificantly decreased in CRC tissues compared with thatin adjacent normal tissues (8.5 ± 2.2 vs. 10.5 ± 2.6, P =0.016, Fig. 2a). These results were further confirmed inthe GSE39582 dataset, in which the relative SPINK4mRNA expression levels in CRC tissues and normaltissues were 8.5 ± 2.6 and 10.1 ± 2.3, respectively (P =0.010, Fig. 2b). To address the sequential expressionchanges from normal colonic mucosa to adenoma tocarcinoma, we analyzed SPINK4 mRNA expression bycomparing 4 pairs of normal colonic mucosa and aden-oma tissues and 4 pairs of adenoma and carcinomatissues from the GSE3880 dataset. The results indicatedthat the relative SPINK4 expression level was decreasedin adenoma compared to that in adjacent normal mu-cosa (4.1 ± 0.1 vs. 4.2 ± 0.1, P = 0.007, Fig. 2d). However,SPINK4 expression was similar between adenoma andcarcinoma (4.1 ± 0.0 vs. 4.2 ± 0.1, P = 0.206, Fig. 2e).

Fig. 2 SPINK4 is downregulated in CRC. SPINK4 expression was significantly decreased in CRC tissues compared with that in paired adjacentnormal tissues at the mRNA level in the (a) GSE32323 dataset and the (b) GSE39582 dataset and at the protein level in (c) our own cohort. dSPINK4 expression was decreased in adenoma tissues compared to that in adjacent normal mucosal tissues in GSE3880. e SPINK4 expression wassimilar between adenoma and carcinoma tissues in GSE3880. Validation of the expression of SPINK4 at the protein level in (f) normal and (g) CRCtissues by the Human Protein Atlas database (IHC staining)

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The mRNA expression of SPINK4 in other cell lines,organs and cancersThe SPINK4 mRNA levels in other various cell lines andnormal organ tissues were analyzed via the IST Onlinedatabase. This analysis revealed that SPINK4 was highlyexpressed in normal colorectal, small intestinal andstomach tissues as well as in gastrointestinal (GI) systemcell lines (Fig. 3). In addition, the differences in SPINK4expression in other tumor and normal tissues ofmultiple cancer types were analyzed in the GEPIA data-base. The results revealed that SPINK4 expression washigher in pancreatic adenocarcinoma (PAAD) and

gastric adenocarcinoma (STAD) tissues than in the cor-responding normal tissues (Fig. 4).

The protein expression of SPINK4 was downregulated inCRC tissuesTo further address the change in SPINK4 proteinexpression in CRC tissues, data mining in the HumanProtein Atlas was first performed. In all 3 normal colontissues, SPINK4 expression was strongly positive, asdetermined by IHC staining, and was mainly located inthe cytoplasm and membrane (Fig. 2f). However, amongthe 12 CRC tissues examined, 7 were negative for

Fig. 3 SPINK4 was highly expressed in normal colorectal, small intestinal and stomach tissues as well as in GI system cell lines, as evidenced byanalysis of the IST Online database. a shows SPINK4 expression at tissue levels of different organs; b shows SPINK4 expression in different celllines. GI system, gastrointestinal system

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SPINK4 staining (negative SPINK4 staining rate: CRCtissues vs normal colon tissues: 58.3% vs 0%, Fig. 2g).Then, IHC staining was used to assess the levels ofthe SPINK4 protein in 81 colorectal cancer tissuescompared with adjacent normal tissues. Positive ex-pression of the SPINK4 protein was found in 98.8%(80/81) of the normal colorectal tissues and 30.9%(25/81) of the CRC tissues (P < 0.001). Moreover, the

SPINK4 protein was expressed at significantly lowerlevels in CRC tissues (total score: 0.3 ± 0.5) than innormal tissues (total score: 2.7 ± 0.6; P = 0.016,Fig. 2c). SPINK4 was mainly located in the cytoplasmand membrane of normal mucosal epithelial cells andprimary cancer cells. Images illustrating differentSPINK4 expression levels in CRC tissues and pairedadjacent normal tissues are shown in Fig. 5.

Fig. 4 SPINK4 expression was higher in pancreatic adenocarcinoma (PAAD) and gastric adenocarcinoma (STAD) tissues than in the correspondingnormal tissues, as evidenced by analysis of the GEPIA database. BLCA, Bladder Urothelial Carcinoma; BRCA, Breast invasive carcinoma; CESC,Cervical squamous cell carcinoma and endocervical adenocarcinoma; CHOL, Cholangio carcinoma; COAD, Colon adenocarcinoma; ESCA,Esophageal carcinoma; HNSC, Head and Neck squamous cell carcinoma; KICH, Kidney Chromophobe; KIRC, Kidney renal clear cell carcinoma; KIRP,Kidney renal papillary cell carcinoma; LIHC, Liver hepatocellular carcinoma; LUAD, Lung adenocarcinoma; LUSC, Lung squamous cell carcinoma;PAAD, Pancreatic adenocarcinoma; PCPG, Pheochromocytoma and Paraganglioma; PRAD, Prostate adenocarcinoma; READ, Rectumadenocarcinoma; SARC, Sarcoma; SKCM, Skin Cutaneous Melanoma; STAD, gastric adenocarcinoma; THCA, Thyroid carcinoma; THYM, Thymoma;UCEC, Uterine Corpus Endometrial Carcinoma

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Correlations between SPINK4 expression andclinicopathological characteristicsSubsequently, the correlations between SPINK4 expres-sion and the clinicopathological characteristics ofpatients with CRC were investigated. Low SPINK4 ex-pression was more frequently observed in patients withmore advanced TNM stage (stage III-IV: 33/56, 58.9%)than in patients with lower TNM stage (stage I-II: 8/25,32.0%, P = 0.025). In addition, low SPINK4 expressionwas significantly related to lower BMI (22.0 ± 3.4 vs. 23.9 ±2.5, P = 0.031). SPINK4 expression was not associatedwith tumor grade, since the percentage of well to mod-erately differentiated tumors was similar between thelow SPINK4 expression and high SPINK4 expressiongroups (96.4% vs. 92.0%, P = 0.583). No correlationswere observed for gender, age, tumor size, histologicaltype, pretreatment CEA level, pretreatment CA199

level, perineural invasion status, venous invasion status,or tumor location (Table 1).

Correlations between SPINK4 expression and CRC patientsurvivalThe prognostic significance of SPINK4 in CRC patientswas explored first by a data mining approach in theGEO and TCGA databases at the mRNA level. Thepatient characteristics of each study cohort are summa-rized in Table 2. The study cohorts were divided intotwo groups according to the cutoff points, which wereassessed using X-tile. CRC patients from the GSE24551dataset with low SPINK4 mRNA levels had significantlylower 5-year overall survival rates (5Y-OS) than thosewith high SPINK4 mRNA levels (56.2% vs. 78.9%, P =0.022, Fig. 6a). In the GSE39582 dataset, low SPINK4mRNA expression was also significantly associated with

Fig. 5 Representative images of SPINK4 immunohistochemical staining in CRC tissues from our study cohort. a Negative SPINK4 expression inCRC tissue (magnification × 200); b low SPINK4 expression in CRC tissue (magnification × 200); c moderate-strong SPINK4 expression in CRC tissue(magnification × 200); d strong SPINK4 expression in normal tissue. (magnification × 200); e Representative images of SPINK4 protein expression insections of nonneoplastic mucosa adjacent to tumors (magnification × 200); f Representative images of SPINK4 protein expression in sections ofnonneoplastic mucosa adjacent to tumors (magnification × 400)

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decreased 5Y-OS in CRC patients (61.4% vs. 72.0%, P =0.022, Fig. 6b). These results were also confirmed in pa-tients from the TCGA database (5Y-OS of low expressionvs. high expression: 38.5% vs. 76.9%, P<0.001, Fig. 6c). Atthe protein level, IHC staining was carried out in our owncohort, and survival analysis revealed that CRC patientswith low levels of SPINK4 protein expression had signifi-cantly worse disease-free survival (DFS) and overall survivalthan those with high levels of SPINK4 protein expression(5Y-DFS of low expression vs. high expression: 58.5% vs.83.6%, P = 0.007, Fig. 6d; 5Y-OS of low expression vs. highexpression: 58.4% vs. 83.6%, P = 0.014, Fig. 6e). Further-more, SPINK4 was confirmed as an independent indicatorof poor survival in patients with CRC in multivariate Coxproportional hazard regression in all 3 databases(GSE24551, HR = 0.462, P = 0.056; GSE39582, HR = 0.636,P = 0.014; TCGA database, HR = 0.301, P = 0.014) and inour own cohort (HR = 0.299, P = 0.027 for OS; HR = 0.264,P = 0.014 for DFS) (Tables 3-4). In addition, other inde-pendent factors included TNM stage (GSE24551,GSE39582, TCGA, our cohort), age (GSE39582, TCGA,our cohort), microsatellite status (GSE24551), KRAS status(GSE39582), perineural invasion status (TCGA) and ven-ous invasion status (TCGA).

The potential functions of SPINK4 in CRCTo investigate the function of SPINK4 in CRC, single-cellsequencing data from GSE81861 were analyzed at the

single-cell level. The relationships between SPINK4 expres-sion and the 14 cellular functional states were evaluated bylinear correlation analysis (Additional file 1: Figure S1A).The results showed that SPINK4 is significantly positivelycorrelated with cell differentiation (r2 = 0.446, P = 0.002;Additional file 1: Figure S1B) and inflammation (r2 = 0.543,P < 0.001; Additional file 1: Figure S1C) but significantlynegatively correlated with cell DNA repair (r2 = − 0.433,P = 0.003; Additional file 1: Figure S1D) and stemness(r2 = − 0.556, P < 0.001; Additional file 1: Figure S1E).Then, GSEA was conducted by analyzing data from theGSE24551 dataset and GSE39582 dataset. Nine hallmarkgene sets, including “OXIDATIVE PHOSPHORYLATION”,“INOSITOL PHOSPHATE METABOLISM”, “ALZHEI-MER’S DISEASE”, “MELANOGENESIS”, “PARKINSON’SDISEASE”, “FRUCTOSE AND MANNOSE METABOL-ISM”, “BUTANOATE METABOLISM”, “AMINO SUGARAND NUCLEOTIDE SUGAR METABOLISM” and“PHOSPHATIDYLINOSITOL SIGNALING SYSTEM”,were enriched and shared by both GSE datasets andare suspected to be the crucial signatures of highSPINK4 expression (Additional file 3: Table S1,Additional file 2: Figure S2).

DiscussionThe family of SPINK protease inhibitors originally con-sisted of four members in humans: SPINK1, SPINK2,SPINK4, and SPINK5 [8]. SPINK1 is mainly produced in

Table 1 Correlations between SPINK4 expression and the clinicopathological features of patients with CRC in our own study cohort

Low expression High expression P value

Gender 0.405

Male 28 (50.0) 15 (60.0)

Female 28 (50.0) 10 (40.0)

Age (years) 63.3 ± 12.4 64.1 ± 12.1 0.809

BMI (kg/m2) 22.0 ± 3.4 23.9 ± 2.5 0.031

TNM stage 0.025

I-II 23 (41.1) 17 (68.0)

III-IV 33 (58.9) 8 (32.0)

Tumor size (cm) 5.0 ± 2.0 4.1 ± 1.6 0.075

Histological type 0.366

Adenocarcinoma 53 (94.6) 22 (88.0)

Mucinous adenocarcinoma 3 (5.4) 3 (12.0) 0.306

Pretreatment CEA level (ng/mL) 11.1 ± 22.1 11.4 ± 22.1 0.953

Pretreatment CA199 level (U/ml) 32.8 ± 75.3 17.5 ± 17.9 0.319

Perineural invasion 4 (7.1) 0 (0.0)

Venus invasion 8 (14.3) 3 (12.0) 1.000

Tumor location 0.874

Left colon 5 (8.9) 3 (12.0)

Right colon 19 (33.9) 9 (36.0)

Rectum 32 (57.1) 13 (52.0)

Tumor grade 0.583

Well to moderately differentiated 54 (96.4) 23 (92.0)

Poorly differentiated 2 (3.6) 2 (8.0)

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pancreatic acinar cells and is expressed in variouscancers and inflammatory states. In addition to being aprotease inhibitor, SPINK1 also acts as an acute-phasereactant and a growth factor. Furthermore, it has beenshown to modulate apoptosis [20]. Ozaki et al. [21] sug-gested that SPINK1 stimulates the proliferation of pan-creatic cancer cells through the EGFR/mitogen-activatedprotein kinase cascade. Ida et al. [22] demonstrated thatSPINK1 stimulates the proliferation of colon cancer cellsand is involved in colorectal cancer progression. More-over, overexpression of SPINK1 is associated withadverse prognosis in other cancers, including prostatecancer [23], hepatocellular cancer [24] and breast cancer[25]. Thus, SPINK1 can be used as a prognostic tumormarker. However, there have been only a few studies onthe gene encoding SPINK4, another member of theSPINK family, in tumors. The analysis in the presentstudy revealed that SPINK4 was highly expressed innormal colorectal, small intestinal and stomach tissuesas well as in GI system cell lines. We first investigatedSPINK4 mRNA expression in tumors in data from theTCGA and two GEO datasets (GSE32323 andGSE39582), and the results showed that SPINK4 mRNAexpression was significantly decreased in CRC tissues

compared with that in paired adjacent normal tissues. Inaddition to being downregulated in CRC, SPINK4 ex-pression was higher in pancreatic adenocarcinoma(PAAD) and gastric adenocarcinoma than in the corre-sponding normal tissues at the RNA level. The changein SPINK4 protein expression in CRC tissues was thenvalidated by data mining of the Human Protein Atlasand by IHC staining in our own samples. Consistentwith the predictive results in the database analysis, theSPINK4 protein was expressed at significantly lowerlevels in the 81 CRC tissues than in the paired normaltissues. Furthermore, SPINK4 mRNA expression wasdecreased in adenoma compared to that in adjacent nor-mal mucosa in the present study. However, although theexpression of SPINK4 in carcinoma tended to be furtherdecreased compared to that in adenoma, the differencewas not statistically significant. These results suggestthat the decrease in SPINK4 expression is an early eventin colon carcinogenesis. Due to the limited sample sizeof adenoma cases in the present study, whether SPINK4can be used as a predictor of CRC formation requiresfurther study. Interestingly, the serum SPINK4 level wasincreased in patients with CRC compared with that inhealthy controls in previous research [10]. Since proteins

Table 2 Clinical characteristics of patients in GSE24551, GSE39582 and TCGA database

Datasets Characteristics Values

GSE24551 dataset TNM stage II 90 (56.3)

III 70 (43.8)

Microsatellite status MSS, MSI-L 126 (78.8)

MSI-H 21 (13.1)

Missing 13 (8.1)

GSE39582 dataset Gender Male 288 (54.5)

Female 240 (45.5)

Age (years) 66.6 ± 13.4

TNM stage 0 4 (0.8)

I 32 (6.1)

II 246 (46.6)

III 188 (35.6)

IV 58 (11.0)

BRAF status WT 431 (81.6)

MT 47 (8.9)

Missing 50 (9.5)

KRAS status WT 305 (57.8)

MT 202 (38.3)

Missing 21 (4.0)

TP53 status WT 148 (28.0)

MT 174 (33.0)

Missing 206 (39.0)

MMR status pMMR 413 (78.2)

dMMR 69 (13.1)

Missing 46 (8.7)

TCGA database Gender Male 234 (53.4)

Female 204 (46.6)

Age (years) 66.6 ± 13.0

TNM stage I 73 (16.7)

II 194 (44.3)

III 99 (22.6)

IV 61 (13.9)

Missing 11 (2.5)

Pretreatment CEA level

(ng/ml) 38.4 ± 201.3

Histological type Adenocarcinoma 373 (85.2)

Mucinous adenocarcinoma 60 (13.7)

Missing 5 (1.1)

Perineural invasion No 131 (29.9)

Yes 45 (10.3)

Missing 262 (59.8)

Tumor deposits No 184 (42.0)

Yes 38 (8.7)

Missing 216 (49.3)

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Table 2 Clinical characteristics of patients in GSE24551, GSE39582 and TCGA database (Continued)

Datasets Characteristics Values

KRAS status WT 24 (5.5)

MT 22 (5.0)

Missing 392 (89.5)

MSS Microsatellite Stable, MSI-L Microsatellite instability-low, MSI-H Microsatellite instability-high, WT Wide type, MT Mutation type, DMMR Mismatch repairdeficient, pMMR Mismatch repair proficient

Fig. 6 Low SPINK4 levels were associated with significantly decreased overall survival in CRC patients from a GSE24551, b GSE39582, c TCGA and dour study cohort. Low SPINK4 levels were associated with significantly decreased disease-free survival rates in CRC patients from e our study cohort

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Table 3 Multivariate analysis of indicators for overall survival in CRC patients in GSE24551, GSE39582 and TCGA database

Datasets Variable Group HR (95% CI) P value

GSE24551 dataset Univariate analysis

TNM stage (III vs. II) 1.966 1.190 3.246 0.008

Microsatellite status (MSI-H vs. MSS, MSI-L) 0.246 0.077 0.786 0.018

SPINK4 expression (high vs. low) 0.413 0.188 0.906 0.027

Multivariate analysis

TNM stage (III vs. II) 1.977 1.181 3.310 0.010

Microsatellite status (MSI-H vs. MSS, MSI-L) 0.231 0.072 0.738 0.013

SPINK4 expression (high vs. low) 0.462 0.210 1.018 0.056

GSE39582 dataset Univariate analysis

Gender (female vs. male) 0.773 0.569 1.051 0.100

Age (per year) 1.024 1.011 1.037 0.000

TNM stage (III-IV vs. I-II) 2.033 1.644 2.513 0.000

MMR status (dMMR vs. pMMR) 0.808 0.488 1.340 0.410

TP53 status (MT vs. WT) 1.290 0.884 1.883 0.187

KRAS status (MT vs. WT) 1.347 0.989 1.834 0.059

BRAF status (MT vs. WT) 1.162 0.681 1.982 0.582

SPINK4 expression (high vs. low) 0.662 0.464 0.944 0.023

Multivariate analysis

Age (per year) 1.025 1.012 1.039 0.000

TNM stage (III-IV vs. I-II) 1.921 1.406 2.625 0.000

KRAS status (MT vs. WT) 1.365 1.001 1.861 0.050

SPINK4 expression (high vs. low) 0.636 0.444 0.911 0.014

TCGA dataset Univariate analysis

Gender (female vs. male) 0.884 0.593 1.318 0.545

Age (per year) 1.018 1.002 1.035 0.030

TNM stage (III-IV vs. I-II) 3.002 1.979 4.554 0.000

KRAS status (MT vs. WT) 1.416 0.610 3.288 0.418

Tumor deposits (yes vs. No) 1.752 0.854 3.594 0.126

Perineural invasion (yes vs. No) 1.938 0.981 3.826 0.057

Venus invasion (yes vs. No) 2.566 1.658 3.970 0.000

Pretreatment CEA level (per ng/mL) 1.001 1.001 1.002 0.000

Lymphatic invasion (yes vs. No) 2.140 1.393 3.287 0.001

Histological type (mucinous adenocarcinoma vs. adenocarcinoma) 1.374 0.802 2.352 0.247

SPINK4 expression (high vs. low) 0.479 0.321 0.714 0.000

Multivariate analysis

Age (per year) 1.040 1.003 1.078 0.034

TNM stage (III-IV vs. I-II) 3.750 1.323 10.635 0.013

Perineural invasion (yes vs. No) 2.359 0.864 6.445 0.094

Venous invasion (yes vs. No) 2.773 1.066 7.217 0.037

SPINK4 expression (high vs. low) 0.301 0.116 0.785 0.014

MSS, microsatellite Stable; MSI-L, microsatellite instability-low; MSI-H, microsatellite instability-high; WT, wide type; MT, mutation type

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are secreted by various cells as a response to variousstimuli, determining whether serum SPINK4 is directlyderived from the tumor needs further research. Previous

cancer research demonstrated a very small overlapbetween malignant differentially expressed proteins inserum and tissue [26].

Table 4 Multivariate analysis of indicators for survival in CRC patients in in our own cohort

Variable Group HR (95% CI) P value

Overall survival

Univariate analysis

Gender (female vs. male) 0.583 0.275 1.236 0.159

Age (per year) 1.037 1.004 1.070 0.026

BMI (per kg/m2) 0.963 0.862 1.074 0.496

TNM stage (per stage) 3.135 1.936 5.078 0.000

Tumor size (per cm) 1.091 0.925 1.287 0.300

Pretreatment CEA level (per ng/mL) 1.014 1.002 1.027 0.024

Pretreatment CA199 level (per U/ml) 1.004 1.001 1.007 0.004

Histological type (mucinous adenocarcinoma vs.adenocarcinoma)

1.994 0.693 5.739 0.201

Perineural invasion (yes vs. No) 3.796 1.133 12.712 0.031

Venus invasion (yes vs. No) 1.398 0.533 3.669 0.496

Tumor location 0.981 0.568 1.696 0.946

Differentiation (well to moderately differentiated vs.poorly differentiated)

1.435 0.341 6.043 0.622

SPINK4 expression (high vs. low) 0.289 0.100 0.832 0.021

Multivariate analysis

Age (per year) 1.034 1.003 1.066 0.031

TNM stage (per stage) 3.093 1.873 5.109 0.000

SPINK4 expression (high vs. low) 0.299 0.103 0.872 0.027

Disease-free survival

Univariate analysis

Gender (female vs. male) 0.616 0.298 1.273 0.191

Age (per year) 1.034 1.003 1.066 0.033

BMI (per kg/m2) 0.962 0.867 1.069 0.474

TNM stage (per stage) 3.104 1.943 4.957 0.000

Tumor size (per cm) 1.099 0.936 1.291 0.247

Pretreatment CEA level (per ng/mL) 1.012 1.000 1.024 0.059

Pretreatment CA199 level (per U/ml) 1.004 1.001 1.007 0.003

Histological type (mucinous adenocarcinoma vs.adenocarcinoma)

1.916 0.670 5.482 0.225

Perineural invasion (yes vs. No) 2.918 0.873 9.757 0.082

Venus invasion (yes vs. No) 1.534 0.629 3.741 0.347

Tumor location 1.051 0.617 1.792 0.855

Differentiation (well to moderately differentiated vs.poorly differentiated)

1.424 0.340 5.975 0.629

SPINK4 expression (high vs. low) 0.261 0.091 0.747 0.012

Multivariate analysis

TNM stage (per stage) 3.298 1.986 5.475 0.000

SPINK4 expression (high vs. low) 0.264 0.091 0.766 0.014

Wang et al. BMC Cancer (2019) 19:1258 Page 12 of 15

With regard to the value of serum SPINK4 for predict-ing survival, Xie et al. [10] did not find that serumSPINK4 was associated with OS or DFS in CRC patients.The short follow-up time (less than 10months in half of

the patients) as well as the small sample size in thatstudy might partially explain the negative result. At thetissue level in the present study, IHC staining in ourown cohort indicated that the expression level of the

Wang et al. BMC Cancer (2019) 19:1258 Page 13 of 15

SPINK4 protein was significantly associated with reducedsurvival rates in patients with CRC. To obtain a reliableconclusion, these results were further externally validatedin 3 other independent databases (TCGA and GSE24551,GSE39582). Additionally, in our multivariate Cox propor-tional hazard regression model, SPINK4 was confirmed asan independent indicator of poor survival in CRC patientsin all 3 databases (GSE24551, GSE39582, TCGA) and inour own cohort. These findings suggest that SPINK4 maybe exploited as a potential novel indicator of poor survivalin patients with CRC.The functional and pathway enrichment analysis of

SPINK4 in CRC showed that biological processes such asoxidative phosphorylation, metabolism of some compo-nents, and process in Alzheimer’s disease were signifi-cantly enriched. In cancer cells, there is enhanced glucoseuse, with the rate of the tricarboxylic acid cycle andoxidative phosphorylation slowed and glycolysis increased,as a way to generate energy [27]. This metabolic switchprovides substrates for cell growth and division and freeenergy. Blocking these metabolic pathways could lead to anew approach in cancer treatment [27]. The gene setsassociated with oxidative phosphorylation-related path-ways were enriched in the samples with high SPINK4expression in the current study. Furthermore, at thesingle-cell level functional analysis in our study, SPINK4was deregulated in cancer stem cells, which were demon-strated to show a distinct metabolic phenotype that can behighly glycolytic or oxidative phosphorylation-dependent[28]. Metabolic pathways, including inositol phosphatemetabolism [29], fructose and mannose metabolism [30],and butanoate metabolism [31], were reported to beassociated with cancer development. In the present study,the SPINK4 expression level was decreased in CRC. How-ever, the process in Alzheimer’s disease pathway wassignificantly related to high expression of SPINK4. BothAlzheimer’s disease and cancer are prevalent in theelderly. Some epidemiological studies have reported anegative association between Alzheimer’s disease and can-cer. The results of a meta-analysis suggested that individ-uals diagnosed with Alzheimer’s disease had a decreasedrisk for incident cancer by 42%, and patients with a historyof cancer had a 37% decreased risk of Alzheimer’s disease[32]. However, the underlying mechanism is still unclear.Several basic studies have indicated that neurodegenera-tive disorders and cancer share several biological pathwaysthat may contribute to this negative association [33]. Forinstance, deletion or mutation of Pin1 can induce Alzhei-mer’s disease-like pathological changes in mice [34]. How-ever, Pin1 is overexpressed and/or activated by multiplemechanisms in many common human cancers and actson multiple signaling pathways to promote tumorigenesis.Inhibition of Pin1 in animal models has profound antitu-mor effects [33]. Moreover, lower SPINK4 expression was

found to be associated with CRC cell stemness propertiesand undifferentiated states in the present study. It was re-ported that HMGA1 promotes cancer stem cell propertiesand plays a role in the pathogenesis of Alzheimer’s disease[35]. In addition, higher DNA repair ability was related tolower SPINK4 levels in the single-cell analysis. This rela-tionship could be explained by the observation that cancerstem cells have increased DNA repair abilities and demon-strate resistance to DNA-damaging treatment approaches[36]. These GSEA data and single-cell functional analysesprovide directions for further research on the mechanismof SPINK4 in the development of CRC.Our study has certain limitations. First, only small CRC

tissue samples were collected retrospectively to investigatethe impact of SPINK4 on the long-term survival of CRCpatients, although the results were further externallyvalidated in 3 other independent databases (TCGA,GSE24551, and GSE39582). More studies are needed toconfirm the results. Second, the exact biological functionof SPINK4 in CRC and its detailed molecular regulationmechanisms were not assessed in the present study. Thehypothesis drawn from GSEA and the single-cell func-tional and sequencing data analyses needs to be furtherconfirmed by in vitro and in vivo experiments.

ConclusionsThis preliminary study confirmed by using multipledatasets and our own database that reduced expressionof SPINK4 relates to poor survival in CRC, functioningas a novel indicator.

Supplementary informationSupplementary information accompanies this paper at https://doi.org/10.1186/s12885-019-6484-5.

Additional file 1: Figure S1. The functional states associated with SPINK4at the single-cell level. (A) The heatmap displays correlations between SPINK4and the functional states of cells: SPINK4 was significantly positively correlatedwith cell differentiation (B) and inflammation (C) and was significantlynegatively correlated with cell DNA repair (D) and stemness (E).

Additional file 2: Figure S2. GSEA identified the most significantbiological processes related to SPINK4 based on GSE39582 and GSE24551.GSE39582 and GSE24551 share 9 gene sets: “OXIDATIVEPHOSPHORYLATION”, “INOSITOL PHOSPHATE METABOLISM”,“ALZHEIMER’S DISEASE”, “MELANOGENESIS”, “PARKINSON’S DISEASE”,“FRUCTOSE AND MANNOSE METABOLISM”, “BUTANOATE METABOLISM”,“AMINO SUGAR AND NUCLEOTIDE SUGAR METABOLISM” and“PHOSPHATIDYLINOSITOL SIGNALING SYSTEM”.

Additional file 3: Table S1. The gene sets that were significantlyassociated with SPINK4 by Gene set enrichment analysis (GSEA)

AbbreviationsCRC: Colorectal cancer; GEO: Gene Expression Omnibus; GSEA: Gene SetEnrichment Analysis; IHC staining: Immunohistochemical staining;SPINK: Serine protease inhibitors, Kazal type; TCGA: The Cancer Genome Atlas

AcknowledgementsNot applicable.

Wang et al. BMC Cancer (2019) 19:1258 Page 14 of 15

Authors’ contributionsXJW and PC conceived and designed the study. XJW, QY, YYZ, WMG, XRL,YH and SHH contributed to the computational analyses and confirmed theresults. YWS, JYL and JL gave advice on the software applications. All authorshave read and approved the final version of this manuscript.

FundingNational Clinical Key Specialty Construction Project (General Surgery) ofChina (No. 2012–649) in the design of the study. National Natural ScienceFoundation of China (81902378), Young Scientist Foundation of FujianProvincial Commission of Health and Family Planning (2017-1-39), theStartup Fund for Scientific Research, and Fujian Medical University (No.2016QH027, No. 2018QH1028) in the collection, analysis, and interpretationof data. Joint Funds for the Innovation of Science and Technology, Fujianprovince (2017Y9038, 2018Y9030, 2017Y9103). Fujian Science andTechnology Project (2016 J01456), and Young and Middle-aged BackboneTraining Project in the Health System of Fujian province (2019-ZQN-45) inwriting the manuscript.

Availability of data and materialsThe dataset supporting the conclusions of this article is included within thearticle and its Additional file 3: Table S1. All TCGA related data can beobtained from the TCGA Data Portal via https://tcga-data.nci.nih.gov/. AllGEO related data can be obtained from the GEO Data Portal via https://www.ncbi.nlm.nih.gov/geo/.

Ethics approval and consent to participateThe study was approved by the ethics committee of the Union Hospital,Fujian Medical University, under registration number 2018KY070. The patientsenrolled all presented written informed consent. Tissue was obtained fromthe pathology lab before being discarded. No identifiers were included andthere was no contact with donor.

Consent for publicationNot applicable.

Competing interestsThe authors declare that they have no competing interests.

Author details1Department of Colorectal Surgery, Union Hospital, Fujian Medical University,29 Xin-Quan Road, Fuzhou, Fujian 350001, People’s Republic of China.2Department of Pathology, Union Hospital, Fujian Medical University, Fuzhou,People’s Republic of China. 3Department of General and GastrointestinalSurgery, Suez Canal University, Suez, Egypt. 4Clinical Laboratory, UnionHospital, Fujian Medical University, Fuzhou, People’s Republic of China.

Received: 4 March 2019 Accepted: 18 December 2019

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